Protein purification using hcic and ion exchange chromatography

ABSTRACT

The present invention provides methods for purifying proteins. In particular, the methods employ a two-step non-affinity chromatography process without the use of an in-process tangential flow filtration step.

RELATED APPLICATION

This application is a continuation of U.S. 371 application Ser. No.11/918,155 filed Dec. 16, 2008, which claims the benefit of U.S.Provisional Application No. 60/670,457 filed Apr. 11, 2005, whosecontents are hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to protein purification using atwo-step non-affinity chromatography process without the use of anin-process tangential flow filtration step. In particular, the inventionrelates to a method for purifying polypeptides (such as recombinantproteins, antibodies, antibody fragments, Fab and Fv related products,single-chain antibodies, diabodies, linear antibodies, bispecificantibodies, multispecific antibodies including those from antibodyfragments, fusion proteins with Fc like regions and antibody-likemolecules, e.g. immunoadhesins) from a composition comprising thepolypeptide and one or more contaminants.

BACKGROUND OF THE INVENTION

The large-scale, economic purification of proteins is an increasinglyimportant problem for the biopharmaceutical industry. Therapeuticproteins are typically produced using prokaryotic or eukaryotic celllines that are engineered to express the protein of interest from arecombinant plasmid containing the gene encoding the protein. Separationof the desired protein from the mixture of components fed to the cellsand cellular by-products to an adequate purity, e.g., sufficient for useas a human therapeutic, poses a formidable challenge to biologicsmanufacturers for several reasons.

Manufacturers of protein-based pharmaceutical products must comply withstrict regulatory standards, including extremely stringent purityrequirements. To ensure safety, regulatory agencies, such as Food andDrug Administration (FDA), require that protein-based pharmaceuticalproducts are substantially free from impurities, including both productrelated contaminants such as aggregates, fragments and variants of therecombinant protein and process related contaminants such as host cellproteins, media components, viruses, DNA and endotoxins. While variousprotein purification schemes are available and widely used in thebiopharmaceutical industry, they typically include anaffinity-purification step, such as Protein A purification in the caseof antibodies, in order to reach a pharmaceutically acceptable degree ofpurity.

Despite the advent of advanced chromatography and filtration methods,affinity chromatography is still often employed as a capture step tomeet the purity, yield, and throughput requirements forbiopharmaceutical antibody purification in order to achieve therapeuticgrade purity. Despite a high binding affinity of Protein Achromatography for antibodies (about 10⁻⁸ M for human IgG), and abilityto remove as much as 99.5% of impurities, affinity chromatography is anexpensive purification step for use in purifying therapeutic proteins ona commercial scale. Not only is Protein A significantly more expensivethan non-affinity media, it also has problems such as resin instability,difficulty with cleaning, ligand leakage, and potential immunogenicityof Protein A or Protein A related compounds contaminating the purifiedproduct. The high cost and instability of affinity media, however,increases the ultimate cost of protein-based therapeutics, particularlythose requiring high doses and/or chronic administration. Chromatographyalone can account for two thirds of downstream processing costs and,with respect to monoclonal antibodies, the resin cost for anaffinity-capture column can overwhelm raw materials cost. (see Rathoreet al, Costing Issues in the Production of Biopharmaceuticals, BioPharmInternational, Feb. 1, 2004).

Even if Protein A affinity chromatography is used, adequate purity isoften not achieved unless several purification steps are combined,thereby further increasing cost and reducing product yield. Sinceantibodies account for an increasingly large percentage of therapeuticbiologics on the market and in development for the treatment of cancer,autoimmune disease, infectious disease, cardiovascular disease, andtransplant rejection, there is a need for a process that can purifyproteins using fewer steps and thus realizing lower cost.

US Pat. Pub. No. 2003/0229212, which is hereby incorporated by referencein its entirety, describes a method for purifying antibodies from amixture containing host cell proteins using non-affinity chromatographypurification steps followed by a high-performance tangential-flowfiltration (HPTFF) step. As determined by the reduction of CHOPs(Chinese Hamster Ovary cell Proteins), that purification processresulted in contaminant levels of about 144,780 ppm CHOPs after cationexchange purification, about 410 ppm CHOPs after anion exchangepurification, and about 17-21 ppm CHOPs after HPTFF purification (finalstep), thereby providing a three-step non-affinity process. Thepurification step of HPTFF, which uses a charged membrane to separateimpurities (without limit to relative size), such as proteins, DNA andendotoxins, and to eliminate protein oligomers and degradation productsfrom the mixture containing the antibodies, was essential to achieve thefinal purity.

Moreover, HPTFF has disadvantages, namely (1) it is an extra step in thedevelopment, optimization and scale-up of the protein therapeutic, whichrequires membrane cleaning, validation, commercial availability oflarge-scale GMP cassettes (which is not yet available for HPTFF), andadditional buffers, equipment and greater process time, and (2) itincreases costs while risking potential loss of product by compromisingantibody integrity (by degradation or aggregation or other molecularchange that affects molecular activity).

Therefore, it would be desirable to obtain high purity of a proteintherapeutic from a two-step, non-affinity process, which is not based onHPTFF, at a reduced cost in comparison with affinity-based purificationand other multi-step purification processes. It would be advantageous ifthe non-affinity purification process could remove host cell proteins,nucleic acids, endotoxins, product-related contaminants, e.g.,aggregated, oxidized, deamidated or degraded forms of the protein, andmedia additives, e.g., lipids, vitamins, insulin, methotrexate, aminoacids, carbon sources such as glucose, etc.

The development of a purification scheme applicable to various types ofproteins, scaleable, controllable, and that employs cheaper, reusableresins will allow its integration into product development at a veryearly stage in overall drug development. This approach to the design ofa purification scheme can minimize costly changes to manufacturingprocesses which may otherwise be necessary later in drug development or,worse, after approval. As the process is scaled-up and approaches GMPproduction conditions, additional inherent complexities arise, includingthose associated with resin packing and buffer preparation. Themanufacturing process, and its capacity, can be improved by simplifyingthe purification scheme by eliminating process steps and maximizingthroughput and productivity, while maintaining the integrity and purityof the molecule that is being purified. Therefore, it would be desirableand advantageous to start with a simple and efficient process that canproduce a drug substance of high quality and safety.

SUMMARY OF THE INVENTION

The present invention is based on the unexpected finding thatpolypeptides and proteins, in particular recombinant proteins such asmonoclonal antibodies, can be purified from a contaminated mixturecomprising the molecule of interest and at least one contaminant, suchas host cell proteins and other materials such as media components, byusing a two-step purification process that does not include an affinitychromatography step or an in-process buffer exchange step (e.g., TFF orHPTFF). Rather, only pH manipulations are necessary from the first stepto the second step. The two-step process of the present invention,therefore, substantially reduces the cost of purifying proteins thattypically rely on affinity chromatography or multi-step methodologies toachieve similarly high levels of protein purity.

In the present invention, proteins are highly purified to a therapeuticgrade using the steps of cation exchange chromatography and hydrophobiccharge induction chromatography, without regard to their order andwithout the use of affinity chromatography, in order to yield ahigh-purity protein composition that contains negligible amounts ofimpurities [e.g., host cell proteins, such as CHOPs in amounts less than100 parts per million (ppm)]. In particular embodiments of theinvention, described in the Example infra., HCP CHOP levels were reducedto less than 20 ppm. It is an advantage of a method of the presentinvention that the chromatography steps are interchangeable, therebyallowing practitioners to tailor the method to their needs. Furthermore,a purification process of the present invention can remove not only hostcell protein of interest, but also nucleic acids, endotoxins,product-related contaminants, such as aggregated, oxidized, deamidatedor degraded forms of the protein, and media additives, e.g., lipids,vitamins, insulin, methotrexate, amino acids, carbon sources such asglucose, etc.

The present invention provides methods for the purification ofrecombinant proteins including, but not limited to antibodies, fusionproteins with Fc like regions, and antibody-like molecules, e.g.immunoadhesins, in order to achieve a highly pure protein compositionsuitable for use in preparing therapeutic grade compositions. As aresult, it is an advantage of a method of the present invention that thehighly pure protein composition is suitable for use in preparingtherapeutic grade material and can be used directly in human therapy.

Thus, in one embodiment, a method of the invention is directed topurifying a mixture that contains a target protein and one or morecontaminants by (a) subjecting the mixture to an ion exchangepurification step and a hydrophobic charge induction purification step,where there is no in-process tangential flow filtration step, and (b)isolating the target protein.

In a particular embodiment, the method is directed to purifying amixture that contains a target protein and one or more contaminants suchas host cell proteins (e.g., CHOPs) and nucleic acids by subjecting themixture to cation exchange chromatography and to hydrophobic chargeinduction chromatography, and isolating the target protein to a purityof 100 parts per million (ppm) or less of host cell protein and 10 pg/mgor less of nucleic acids.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows a flow diagram of a two-step purification process of theinvention, where Panel A and Panel B refer to the same resins used indifferent sequential order. Panel A represents a first embodiment of thepurification process of the invention, where cation exchangechromatography (CEXC) is the capture chromatography step followed byhydrophobic charge induction chromatography (HCIC) with only a pHmanipulation in between the two chromatography steps. Panel B representsa second embodiment, where HCIC is the capture chromatography stepfollowed by CEXC with only a pH manipulation in between the twochromatography steps. The approximate binding pH during capture was 6.2for Panel A and 7.0 for Panel B.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, “protein” refers generally to peptides and proteinstypically having at least 5 amino acids or more which are linkedtogether by peptide bonds. A protein can be an antibody, receptor,ligand fusion protein (which comprise at least a portion of two or morepolypeptides that are not fused in their natural state), etc., e.g., seeUS2003022921 and US 20030166869 which are hereby incorporated byreference in their entireties and which list various proteins that canbe purified according to a method of the invention. Polypeptides can befrom any organism (prokaryotic or eukaryotic), particularly frommammals. In addition, a protein or polypeptide purified according to amethod of the invention can be an antibody, fragment, or a variantthereof.

The term “antibody” is used in the broadest sense to cover monoclonalantibodies (including full length monoclonal antibodies), polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies),immunoadhesins and antibody fragments so long as they retain, or aremodified to comprise, a ligand-specific binding domain. An antibody canbe directed against an “antigen” of interest, such as a polypeptide,which may be a biologically relevant therapeutic target, or anon-polypeptide antigen (e.g, such as tumor-associated glycolipidantigens; see U.S. Pat. No. 5,091,178). Preferably, the antigen is abiologically important polypeptide and administration of the antibody toa mammal suffering from a disease or disorder can result in atherapeutic benefit in that mammal Polypeptide antigens includetransmembrane molecules (e.g. receptor) and ligands such as growthfactors. Exemplary antigens include those polypeptides discussed above.The preparation of antigens for generating antibodies and antibodyproduction are well known in the art. Soluble antigens, or fragmentsthereof optionally conjugated to other molecules, can be used asimmunogens for generating antibodies. For transmembrane molecules, suchas receptors, fragments of these (e.g. the extracellular domain of areceptor) can be used as the immunogen. Alternatively, cells expressingthe transmembrane molecule can be used as the immunogen. Such cells canbe derived from a natural source (e.g. cancer cell lines) or may becells which have been transformed by recombinant techniques to expressthe transmembrane molecule.

An “antibody fragment” includes at least a portion of a full lengthantibody and typically an antigen binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; single-chain antibody molecules; diabodies; linearantibodies; and multispecific antibodies formed from antibody fragments.

The term “monoclonal antibody” is used in the conventional sense torefer to an antibody obtained from a population of substantiallyhomogeneous antibodies such that the individual antibodies comprisingthe population are identical except for possible naturally occurringmutations that may be present in minor amounts. Monoclonal antibodiesare highly specific, being directed against a single antigenic site.This is in contrast with polyclonal antibody preparations whichtypically include varied antibodies directed against differentdeterminants (epitopes) of an antigen, whereas monoclonal antibodies aredirected against a single determinant on the antigen. The term“monoclonal”, in describing antibodies, indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, monoclonal antibodiesused in the present invention can be produced using conventionalhybridoma technology first described by Kohler et al., Nature 256:495(1975), or they can be made using recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). Monoclonal antibodies can also be isolatedfrom phage antibody libraries, e.g., using the techniques described inClackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol.222:581-597 (1991); and U.S. Pat. Nos. 5,223,409; 5,403,484; 5,571,698;5,427,908 5,580,717; 5,969,108; 6,172,197; 5,885,793; 6,521,404;6,544,731; 6,555,313; 6,582,915; and 6,593,081).

The monoclonal antibodies described herein include “chimeric” and“humanized” antibodies in which a portion of the heavy and/or lightchain is identical with or homologous to corresponding sequences inantibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which the hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.In some instances, Fv framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature 321:522-525 (1986); Riechmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992).

Chimeric or humanized antibodies can be prepared based on the sequenceof a murine monoclonal antibody prepared as described above. DNAencoding the heavy and light chain immunoglobulins can be obtained fromthe murine hybridoma of interest and engineered to contain non-murine(e.g., human) immunoglobulin sequences using standard molecular biologytechniques. For example, to create a chimeric antibody, the murinevariable regions can be linked to human constant regions using methodsknown in the art (see e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.).To create a humanized antibody, the murine CDR regions can be insertedinto a human framework using methods known in the art (see e.g., U.S.Pat. No. 5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089;5,693,762 and 6,180,370 to Queen et al.).

The monoclonal antibodies described herein also include “human”antibodies, which can be isolated from various sources, including, e.g.,from the blood of a human patient or recombinantly prepared usingtransgenic animals. Examples of such transgenic animals includeKM-Mouse® (Medarex, Inc., Princeton, N.J.) which has a human heavy chaintransgene and a human light chain transchromosome (see WO 02/43478),Xenomouse® (Abgenix, Inc., Fremont Calif.; described in, e.g., U.S. Pat.Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963 toKucherlapati et al.), and HuMAb-Mouse® (Medarex, Inc.; described in,e.g., Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295;Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon etal. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993)Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830;Tuaillon et al., (1994) J. Immunol. 152:2912-2920; Taylor, L. et al.(1994) International Immunology 6: 579-591; and Fishwild, D. et al.(1996) Nature Biotechnology 14: 845-851, U.S. Pat. Nos. 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016;5,814,318; 5,874,299; and 5,770,429; 5,545,807; and PCT Publication Nos.WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO99/45962, WO 01/14424 to Korman et al.). Human monoclonal antibodies ofthe invention can also be prepared using SCID mice into which humanimmune cells have been reconstituted such that a human antibody responsecan be generated upon immunization. Such mice are described in, forexample, U.S. Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.

The term “hypervariable region” is used to describe the amino acidresidues of an antibody which are responsible for antigen-binding. Thehypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; seeKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (i.e. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “Framework”or “FR” residues are those variable domain residues other than thehypervariable region residues.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the “binding domain” of a heterologous “adhesin”protein (e.g. a receptor, ligand or enzyme) with the effector functionsof an immunoglobulin constant domain. Structurally, an immunoadhesincomprises a fusion of the adhesin amino acid sequence with the desiredbinding specificity which is other than the antigen recognition andbinding site (antigen combining site) of an antibody (i.e. is“heterologous”) and an immunoglobulin constant domain sequence. Theimmunoglobulin constant domain sequence in the immunoadhesin ispreferably derived from γ1, γ2, or γ4 heavy chains, since immunoadhesinscomprising these regions can be purified by Protein A chromatography(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)) Immunoadhesins canbe purified according to methods of the present invention.

The term “ligand binding domain” refers to any native cell-surfacereceptor or any region or derivative thereof retaining at least aqualitative ligand binding of a corresponding native receptor. In aspecific embodiment, the receptor is from a cell-surface polypeptidehaving an extracellular domain which is homologous to a member of theimmunoglobulin super gene family. Other receptors, which are not membersof the immunoglobulin super gene family but are nonetheless specificallycovered by this definition, are receptors for cytokines, and inparticular receptors with tyrosine kinase activity (receptor tyrosinekinases), members of the hematopoietin and nerve growth factor receptorsuperfamilies, and cell adhesion molecules, e.g. (E-, L- and P-)selectins.

The term “receptor binding domain” is used to designate any nativeligand for a receptor, including cell adhesion molecules, or any regionor derivative of such native ligand retaining at least a qualitativereceptor binding ability of a corresponding native ligand. Thisdefinition, among others, specifically includes binding sequences fromligands for the above-mentioned receptors.

An “antibody-immunoadhesin chimera” comprises a molecule which combinesat least one binding domain of an antibody (as herein defined) with atleast one immunoadhesin (as defined in this application). Exemplaryantibody-immunoadhesin chimeras are the bispecific CD4-IgG chimerasdescribed in Berg et al., PNAS (USA) 88:4723-4727 (1991) and Chamow etal., J. Immunol. 153:4268 (1994).

As used herein, a “mixture” comprises a polypeptide of interest (forwhich purification is desired) and one or more contaminant, i.e.,impurities. The mixture can be obtained directly from a host cell ororganism producing the polypeptide. Without intending to be limiting,examples of mixtures that can be purified according to a method of thepresent invention include harvested cell culture fluid, cell culturesupernatant and conditioned cell culture supernatant. A mixture that hasbeen “partially purified” has already been subjected to a chromatographystep, e.g., non-affinity chromatography, affinity chromatography, etc. A“conditioned mixture” is a mixture, e.g., a cell culture supernatantthat has been prepared for a chromatography step used in a method of theinvention by subjecting the mixture to one or more of buffer exchange,dilution, salt addition, pH titration or filtration in order to set thepH and/or conductivity range and/or buffer matrix to achieve a desiredchromatography performance. A “conditioned mixture” can be used tostandardize loading conditions onto the first chromatography column. Ingeneral, a mixture can be obtained through various separation means wellknown in the art, e.g., by physically separating dead and viable cellsfrom other components in the broth at the end of a bioreactor run usingfiltration or centrifugation, or by concentration and/or diafiltrationof the cell culture supernatant into specific ranges of pH, conductivityand buffer species concentration.

The terms “impurity” and “contaminant”, and grammatical variationsthereof, are used interchangeably to mean any material, other than theprotein of interest for which it is desirable to have removed from acomposition containing the protein of interest. Contaminants include,but are not limited to, any biological macromolecule such as host cellproteins (e.g., CHOPs), polypeptides other than the protein of interest,nucleic acids (e.g., DNA and RNA), lipids, saccharides, endotoxins,bacteria or other microorganisms such as yeast, media components, andany molecule that is part of an adsorbent used in chromatography whichmay leach into a sample during chromatography, and the like.

The term “protein of interest” and “target protein” are usedinterchangeably to refer to a protein, as described above, e.g., anantibody, for which it is desirable to purify from a mixture accordingto a method of the present invention.

The term “host cell protein”, or “HCP”, refers to any of the proteinsderived from the metabolism (intra and extra-cellular) of the host cellthat expresses the target protein, including any proteins expressed fromthe genome of the host cell or proteins that are recombinantlyexpressed, and which are not considered the target protein. The hostcell can be any cell that is capable of expressing the target protein,particularly mammalian (e.g., CHO and murine myeloma cell lines such asNS0), insect bacterial, plant and yeast cell lines. In a particularembodiment of the invention, the HCP is a “Chinese hamster ovary cellprotein”, or “CHOP”, which refers to any of the host cell proteins(“HCP”) derived from a Chinese hamster ovary (“CHO”) cell culture. TheHCP is present generally as an impurity in a cell culture medium orlysate [(e.g., a harvested cell culture fluid (“HCCF”)], which containsthe protein of interest. The amount of HCP present in a mixturecomprising a protein of interest provides a measure of the degree ofpurity for the protein of interest. Typically, the amount of HCP in aprotein mixture is expressed in parts per million relative to the amountof the protein of interest in the mixture.

The term “parts per million” or “ppm” are used interchangeably herein torefer to a measure of purity of the protein of interest purified by amethod of the invention. The units ppm refer to the amount of HCP innanograms/milliliter per protein of interest in milligrams/milliliter,where the proteins are in solution [i.e., as described in an Exampleinfra, HCP ppm=(CHOP ng/ml)/(protein of interest mg/ml)]. Where theproteins are dried, such as by lyophilization, ppm refers to (HCPng)/(protein of interest mg).

The term “purify”, and grammatical variations thereof, is used to meanthe removal, whether completely or partially, of at least one impurityfrom a mixture containing the polypeptide and one or more impurities,which thereby improves the level of purity of the polypeptide in thecomposition (i.e., by decreasing the amount (ppm) of impurity(ies) inthe composition). According to the present invention, purification isperformed with two non-affinity chromatography steps and no in-processTFF steps. A method of the present invention can purify the protein ofinterest to achieve a composition which contains less than 100 ppm HCPand preferably less than 90 ppm, less than 80 ppm, less than 70 ppm,less than 60 ppm, less than 50 ppm, less than 40 ppm, less than 30 ppm,less than 20 ppm, less than 10 ppm, or less than 5 ppm HCP, asdetermined by ELISA.

As used herein, the term “isolate”, and grammatical variations thereof,refers to the separation of a purified protein of interest fromadditional substances, e.g., from a column or resin used to purify theprotein of interest, in order to achieve a homogenous composition, whichcontains the protein of interest substantially free from contaminants,impurities and other substances.

The term “chromatography” refers to the process by which a solute ofinterest, e.g., a protein of interest, in a mixture is separated fromother solutes in the mixture by percolation of the mixture through anadsorbent, which adsorbs or retains a solute more or less strongly dueto properties of the solute, such as pI, hydrophobicity, size andstructure, under particular buffering conditions of the process.

An “adsorbent” is any solid and fixed substance capable of adsorbinganother substance to its surface by adhesion by either directinteraction with the molecule of interest or interaction with compoundsthat are attached to the adsorbent. Adsorbents that are useful invarious types of chromatography are well known in the art and arereadily available through commercial sources.

The term “affinity chromatography” and “protein affinity chromatography”are used interchangeably to refer to a protein separation technique inwhich a protein of interest is reversibly and specifically bound to abiospecific ligand, usually as a combination of spatial complementarityand one or more types of chemical interactions, e.g., electrostaticforces, hydrogen bonding, hydrophobic forces, and/or van der Waalsforces at the binding site. These interactions are not due to thegeneral properties of the molecule such as isoelectric point,hydrophobicity or size but are a result of specific interactions fromthe molecule of interest and the ligand such as the hydrophobic andprecise protein domain fit for protein A and antibody interactions, forexample. Protein A is an example of an adsorbent, which can be fixed toa solid support, e.g., Sepharose, for binding molecules that contain anF_(C) region. See Ostrove (1990) in Guide to Protein Purification,Methods of Enzymology 182: 357-379, which is incorporated herein byreference in its entirety.

Any ligand can be used to purify its respective specific bindingprotein. Preferably, the biospecific ligand is covalently attached to achromatographic solid phase material and is accessible to the protein ofinterest (e.g. antibody, enzyme, or receptor protein) in solution as thesolution contacts the chromatographic solid phase material. The proteinof interest retains its specific binding affinity for the biospecificligand (antigen, substrate, cofactor, or hormone, for example) duringthe chromatographic steps, while other solutes and/or proteins in themixture do not bind appreciably or specifically to the ligand. Bindingof the protein of interest to the immobilized ligand allowscontaminating proteins or protein impurities to be passed through thechromatographic medium while the protein of interest remainsspecifically bound to the immobilized ligand on the solid phasematerial. The specifically bound protein is then removed form theimmobilized ligand with low pH, high pH, low salt, high salt, competingligand, or the like, and passes through the chromatographic column withthe elution buffer. Contaminating proteins having a lower relativeconcentration to the protein of interest, and other types ofcontaminants such as nucleic acids and endotoxin, that were earlierallowed to pass through the column, may also be present.

The terms “specific binding” and “binding specificity”, and grammaticalvariations thereof, describe the generally specific and reversibleinteractions between a protein of interest and a ligand requiring thecombined effects of spatial complementarity of protein and ligandstructures at a binding site coupled with one or more type ofelectrostatic forces, hydrogen bonding, hydrophobic forces, and/or vander Waals forces at the binding site. The ligand should have chemicallymodifiable groups which allow it to be attached to the matrix withoutdestroying its binding activity. The ligand should ideally have anaffinity for the binding substance in the range 10 to 10⁻⁸M in freesolution. The greater the spatial complementarity and the stronger theother forces at the binding site, the greater will be the bindingspecificity of a protein for its respective ligand. Non-limitingexamples of specific binding include antibody-antigen binding,enzyme-substrate binding, enzyme-cofactor binding, metal ion chelation,DNA binding protein-DNA binding, regulatory protein-proteininteractions, and the like.

The terms “non-affinity chromatography” and “non-affinity purification”refer to a purification step which does not use affinity chromatographybut rather requires a non-specific binding interaction between a solute(e.g., protein of interest) and the adsorbent matrix.

The term “non-specific binding” as used herein, refers to theinteractions between a protein of interest and a ligand or othercompound bound to a solid phase matrix through non-specificinteractions, e.g., through electrostatic forces, hydrogen bonding,hydrophobic forces, and/or van der Waals forces at an interaction site,but lacking the structural complementarity that enhances the effects ofthe non-structural forces such as in affinity (specific) binding.Examples of chromatography processes that rely on non-specific binding,rather than affinity, include ionic exchange chromatography (e.g.,anionic and cationic exchange) and hydrophobic charge inductionchromatography.

The term “hydrophobic charge induction chromatography” (or “HCIC”) is atype of mixed mode chromatographic process in which the protein ofinterest in the mixture binds to a dual mode (i.e., there is one modefor binding and another mode for elution), ionizable ligand [seeBoschetti et al., 2000, Genetic Engineering News 20(13)] through mildhydrophobic interactions in the absence of added salts (e.g. a lyotropicsalts). A “hydrophobic charge induction chromatography resin” is a solidphase that contains a ligand which has the combined properties ofthiophilic effect (i.e., utilizing the properties of thiophilicchromatography), hydrophobicity and an ionizable group for itsseparation capability. Thus, an HCIC resin used in a method of theinvention contains a ligand that is ionizable and mildly hydrophobic atneutral (physiological) or slightly acidic pH, e.g., about pH 5 to 10,preferably about pH 6 to 9.5. At this pH range, the ligand ispredominantly uncharged and binds a protein of interest via mildnon-specific hydrophobic interaction. As pH is reduced, the ligandacquires charge and hydrophobic binding is disrupted by electrostaticcharge repulsion towards the solute due to the pH shift.

Examples of suitable ligands for use in HCIC include any ionizablearomatic or heterocyclic structure (e.g. those having a pyridinestructure, such as 2-aminomethylpyridine, 3-aminomethylpyridine and4-aminomethylpyridine, 2-mercaptopyridine, 4-mercaptopyridine or4-mercaptoethylpyridine, mercaptoacids, mercaptoalcohols, imidazolylbased, mercaptomethylimidazole, 2-mercaptobenzimidazole,aminomethylbenzimidazole, histamine, mercaptobenzimidazole,diethylaminopropylamine, aminopropylmorpholine, aminopropylimidazole,aminocaproic acid, nitrohydroxybenzoic acid, nitrotyrosine/ethanolamine,dichlorosalicylic acid, dibromotyramine, chlorohydroxyphenylacetic acid,hydroxyphenylacetic acid, tyramine, thiophenol, glutathione, bisulphate,and dyes, including derivatives thereof; see Burton and Harding, Journalof Chromatography A 814: 81-81 (1998) and Boschetti, Journal ofBiochemical and Biophysical Methods 49: 361-389 (2001), which are herebyincorporated by reference in their entireties), which has an aliphaticchain and at least one sulfur atom on the linker arm and/or ligandstructure. An example of an HCIC resin includes MEP HYPERCEL (PallCorporation; East Hills, N.Y.).

The terms “ion-exchange” and “ion-exchange chromatography” refer to achromatographic process in which an ionizable solute of interest (e.g.,a protein of interest in a mixture) interacts with an oppositely chargedligand linked (e.g., by covalent attachment) to a solid phase ionexchange material under appropriate conditions of pH and conductivity,such that the solute of interest interacts non-specifically with thecharged compound more or less than the solute impurities or contaminantsin the mixture. The contaminating solutes in the mixture can be washedfrom a column of the ion exchange material or are bound to or excludedfrom the resin, faster or slower than the solute of interest.“Ion-exchange chromatography” specifically includes cation exchange,anion exchange, and mixed mode chromatographies.

The phrase “ion exchange material” refers to a solid phase that isnegatively charged (i.e. a cation exchange resin) or positively charged(i.e. an anion exchange resin). In one embodiment, the charge can beprovided by attaching one or more charged ligands (or adsorbents) to thesolid phase, e.g. by covalent linking. Alternatively, or in addition,the charge can be an inherent property of the solid phase (e.g. as isthe case for silica, which has an overall negative charge).

A “cation exchange resin” refers to a solid phase which is negativelycharged, and which has free cations for exchange with cations in anaqueous solution passed over or through the solid phase. Any negativelycharged ligand attached to the solid phase suitable to form the cationexchange resin can be used, e.g., a carboxylate, sulfonate and others asdescribed below. Commercially available cation exchange resins include,but are not limited to, for example, those having a sulfonate basedgroup (e.g., MonoS, MiniS, Source 15S and 30S, SP Sepharose Fast Flow™,SP Sepharose High Performance from GE Healthcare, Toyopearl SP-650S andSP-650M from Tosoh, Macro-Prep High S from BioRad, Ceramic HyperD S,Trisacryl M and LS SP and Spherodex LS SP from Pall Technologies); asulfoethyl based group (e.g., Fractogel SE, from EMD, Poros S-10 andS-20 from Applied Biosystems); a sulphopropyl based group (e.g., TSK GelSP 5PW and SP-5PW-HR from Tosoh, Poros HS-20 and HS 50 from AppliedBiosystems); a sulfoisobutyl based group (e.g., (Fractogel EMD SO₃ ⁻from EMD); a sulfoxyethyl based group (e.g., SE52, SE53 and Express-IonS from Whatman), a carboxymethyl based group (e.g., CM Sepharose FastFlow from GE Healthcare, Hydrocell CM from Biochrom Labs Inc.,Macro-Prep CM from BioRad, Ceramic HyperD CM, Trisacryl M CM, TrisacrylLS CM, from Pall Technologies, Matrx Cellufine C500 and C200 fromMillipore, CM52, CM32, CM23 and Express-Ion C from Whatman, ToyopearlCM-650S, CM-650M and CM-650C from Tosoh); sulfonic and carboxylic acidbased groups (e.g. BAKERBOND Carboxy-Sulfon from J. T. Baker); acarboxylic acid based group (e.g., WP CBX from J. T Baker, DOWEX MAC-3from Dow Liquid Separations, Amberlite Weak Cation Exchangers, DOWEXWeak Cation Exchanger, and Diaion Weak Cation Exchangers fromSigma-Aldrich and Fractogel EMD COO— from EMD); a sulfonic acid basedgroup (e.g., Hydrocell SP from Biochrom Labs Inc., DOWEX Fine MeshStrong Acid Cation Resin from Dow Liquid Separations, UNOsphere S, WPSulfonic from J. T. Baker, Sartobind S membrane from Sartorius,Amberlite Strong Cation Exchangers, DOWEX Strong Cation and DiaionStrong Cation Exchanger from Sigma-Aldrich); and a orthophosphate basedgroup (e.g., P11 from Whatman).

An “anion exchange resin” refers to a solid phase which is positivelycharged, thus having one or more positively charged ligands attachedthereto. Any positively charged ligand attached to the solid phasesuitable to form the anionic exchange resin can be used, such asquaternary amino groups Commercially available anion exchange resinsinclude DEAE cellulose, Poros PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50from Applied Biosystems, Sartobind Q from Sartorius, MonoQ, MiniQ,Source 15Q and 30Q, Q, DEAE and ANX Sepharose Fast Flow, Q Sepharosehigh Performance, QAE SEPHADEX™ and FAST Q SEPHAROSE™ (GE Healthcare),WP PEI, WP DEAM, WP QUAT from J. T. Baker, Hydrocell DEAE and HydrocellQA from Biochrom Labs Inc., UNOsphere Q, Macro-Prep DEAE and Macro-PrepHigh Q from Biorad, Ceramic HyperD Q, ceramic HyperD DEAE, Trisacryl Mand LS DEAE, Spherodex LS DEAE, QMA Spherosil LS, QMA Spherosil M andMustang Q from Pall Technologies, DOWEX Fine Mesh Strong Base Type I andType II Anion Resins and DOWEX MONOSPHER E 77, weak base anion from DowLiquid Separations, Intercept Q membrane, Matrex Cellufine A200, A500,Q500, and Q800, from Millipore, Fractogel EMD TMAE, Fractogel EMD DEAEand Fractogel EMD DMAE from EMD, Amberlite weak strong anion exchangerstype I and II, DOWEX weak and strong anion exchangers type I and II,Diaion weak and strong anion exchangers type I and II, Duolite fromSigma-Aldrich, TSK gel Q and DEAE 5PW and 5PW-HR, Toyopearl SuperQ-6505,650M and 650C, QAE-550C and 650S, DEAE-650M and 650C from Tosoh, QA52,DE23, DE32, DE51, DE52, DE53, Express-Ion D and Express-Ion Q fromWhatman.

A “mixed mode ion exchange resin” refers to a solid phase which iscovalently modified with cationic, anionic, and/or hydrophobic moieties.Examples of mixed mode ion exchange resins include BAKERBOND ABX™ (J. T.Baker; Phillipsburg, N.J.), ceramic hydroxyapatite type I and II andfluoride hydroxyapatite (BioRad; Hercules, Calif.) and MEP and MBIHyperCel (Pall Corporation; East Hills, N.Y.).

The term “thiophilic” refers to the selectivity that proteins have forsulfone groups that lie in close proximity to thioether groups (Porathet al, 1985). “Thiophilic chromatography” also known as “thiophilicadsorption chromatography”, is a type of non-affinity chromatography inwhich a protein of interest, which contains thiophilic regions andaromatic amino residues, bind to a sulphur containing ligand for theisolation of the protein. A thiophilic gel can be prepared by reducingdivinylsulfone (coupled to Sepharose 4B) with β-mercaptoethanol.Thiophilic adsorption chromatography is based on electron donor-acceptorproperties and is distinct from chromatography based on hydrophobicity.Hydrophobic associations and ionic interactions do not occur withthiophilic sorbents since thio-ethylsulfone structures do not possesspronounced hydrophobicity or ionic charges. Examples of commerciallyavailable thiophilic chromatography resins include Fractogel EMD TA(Merck; Rahway, N.J.), Uniflow and Superflow resin (Clontech) and T-Gel(Pierce).

The term “solid phase” is used to mean any non-aqueous matrix to whichone or more ligands can adhere or alternatively, in the case of sizeexclusion chromatography, it can refer to the gel structure of a resin.The solid phase can be any matrix capable of adhering ligands in thismanner, e.g., a purification column, a discontinuous phase of discreteparticles, a membrane, filter, gel, etc. Examples of materials that canbe used to form the solid phase include polysaccharides (such as agaroseand cellulose) and other mechanically stable matrices such as silica(e.g. controlled pore glass), poly(styrenedivinyl)benzene,polyacrylamide, ceramic particles and derivatives of any of these. Thepresent invention is not limited to any particular solid phase materialfor use in a chromatography step, and those having ordinary skill in theart will be able to select appropriate solid phase material for use inthe present invention.

The term “detergent” refers to ionic, zwitterionic and nonionicsurfactants, which are useful for preventing aggregation of proteins andto prevent non-specific interaction or binding of contaminants to theprotein of interest, and can be present in various buffers used in thepresent invention, including sanitization, equilibration, loading,post-load wash(es), elution or strip buffers. In particular embodiments,a detergent is added to a wash buffer. Examples of detergents that canbe used in the invention include, but are not limited to polysorbates(e.g. polysorbates 20 or 80); poloxamers (e.g. poloxamer 188); Triton;sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octylglycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine;lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-,myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-,linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-betaine (e.g. lauroamidopropyl); myristamidopropyl-,palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methylcocoyl-, or disodium methyl oleyl-taurate; MONAQUAT™ series (MonaIndustries, Inc., Paterson, N.J.); Igepal CA-630, Pluronic, Triton,BRIJ, Atlas G2127, Genapol, HECAMEG, LUBROL PX, MEGA, NP, THESIT, TOPPS,CHAPS, CHAPSO, DDMAU, EMPIGEN BB, AWITTERGENT and C12E8. The detergentcan be added in any working buffer and can also be included in the feedcontaining the molecule of interest. Detergents can be present in anyamount suitable for use in a protein purification process, e.g., fromabout 0.001% to about 20% and typically from about 0.01% to about 1%. Ina particular embodiment, polysorbate 80 is used in a wash buffer forCEXC.

A “buffer” used in the present invention is a solution that resistschanges in pH by the addition of acid or base by the action of itsacid-base conjugates components. Various buffers can be employed in amethod of the present invention depending on the desired pH of thebuffer and the particular step in the purification process [see Buffers.A Guide for the Preparation and Use of Buffers in Biological Systems,Gueffroy, D., ed. Calbiochem Corporation (1975)]. Non-limiting examplesof buffer components that can be used to control the pH range desirablefor a method of the invention include acetate, citrate, histidine,phosphate, ammonium buffers such as ammonium acetate, succinate, MES,CHAPS, MOPS, MOPSO, HEPES, Tris, and the like, as well as combinationsof these TRIS-malic acid-NaOH, maleate, chloroacetate, formate,benzoate, propionate, pyridine, piperazine, ADA, PIPES, ACES, BES, TES,tricine, bicine, TAPS, ethanolamine, CHES, CAPS, methylamine,piperidine, O-boric acid, carbonic acid, lactic acid, butaneandioicacid, diethylmalonic acid, glycylglycine, HEPPS, HEPPSO, imidazole,phenol, POPSO, succinate, TAPS, amine-based, benzylamine, trimethyl ordimethyl or ethyl or phenyl amine, ethylenediamine, or mopholineAdditional components (additives) can be present in a buffer as needed,e.g., salts can be used to adjust buffer ionic strength, such as sodiumchloride, sodium sulfate and potassium chloride; and other additivessuch as amino acids (such as glycine and histidine), chaotropes (such asurea), alcohols (such as ethanol, mannitol, glycerol, and benzylalcohol), detergents (see supra.), and sugars (such as sucrose,mannitol, maltose, trehalose, glucose, and fructose). The buffercomponents and additives, and the concentrations used, can varyaccording to the type of chromatography practiced in the invention.

The pH and conductivity of the buffers can vary depending on which stepin the purification process the buffer is used. In CEXC the pH of thebuffer can be between 3 and 10, more preferably from about pH 4.0 to8.0; conductivity can be from about 0.1 to 40 mS/cm, more preferablyfrom about conductivity 0.5 to 15 mS/cm, depending on the purificationstep and the buffer employed. In HCIC the pH of the buffers can be fromabout 3 to 10, more preferably from about pH 4.0 to 9.0; conductivitycan be from about 0.0 (WFI; water for injection) to 90.0 mS/cm, morepreferably from about 0.1 to 9.0 mS/cm, depending on the purificationstep and the buffer employed. Buffers used throughout the purificationprocess are described in greater detail below for each chromatographystep.

A “sanitization” solution is typically used to clean the resin byremoving any bound contaminants, e.g., those of biological origin, priorto the purification process. Any desirable buffer could be used for thispurpose provided it is compatible with the particular column and resinselected according to a method of the invention. Preferably, the pH ofthe sanitization solution is high, e.g., pH 10 or greater, morepreferably pH 11 or greater, and still more preferably pH 12 or greater;alternatively, the pH of the sanitization solution can be low, e.g. pH 4or less, more preferably pH 3 or less. In a particular embodiment, theHCIC and CEXC columns are cleaned using a sanitization solution thatincludes 1N NaOH, pH>12.

An “equilibration buffer” is used to adjust the pH and conductivity ofthe chromatography column prior to loading the column with the mixturecontaining the protein of interest for purification. Suitable buffersthat can be used for this purpose are well known in the art, e.g., suchas buffers described above, and include any buffer at pH that iscompatible with the selected resin used in the chromatography step forpurifying the protein of interest. This buffer is used to load themixture comprising the polypeptide of interest. The equilibration bufferhas a conductivity and/or pH such that the polypeptide of interest isbound to the resin or such that the protein of interest flows throughthe column while one or more impurities bind to the column. In apreferred embodiment, equilibration of the chromatography column iscompleted when the pH and conductivity are within ±0.2 and ±0.4 mS/cm ofthe equilibrating buffer, respectively, more preferably within ±0.1 and±0.2 mS/cm of the equilibrating buffer, respectively. In preferredembodiments, the equilibration buffer species for CEXC and HCIC aresodium phosphate based. In CEXC the pH of the equilibration buffer isfrom about 3 to about 9, more preferably pH from about 4.0 to 8.0,conductivity from about 0.1 to about 40 mS/cm, more preferably fromabout 0.5 to 10.0 mS/cm. In HCIC the pH of the equilibration buffer isfrom about 5 to about 10, more preferably pH from about 6 to 9,conductivity from about 0.0 (WFI) to about 90 mS/cm, more preferablyconductivity from about 2.0 to 9 mS/cm.

A “loading buffer” is used to load the mixture containing the protein ofinterest onto the column. Any appropriate solution can be used as theloading buffer. The conductivity and pH of the loading buffer in thepresent process is selected such that the protein of interest is boundto the resin while contaminants are able to flow through the column.Preferably, the loading buffer can be buffer exchanged. The loadingbuffer can also be prepared from a buffered mixture derived from aprevious purification step, such as the elution buffer. Suitable buffersfor use as a loading buffer with the selected resin are well known inthe art, e.g., such as those described above. It shall be appreciated bythose having ordinary skill in the art that loading buffers for CEXC andHCIC can be used at comparable (if not the same) pH and conductivitiesas described above for the equilibration buffers for CEXC and HCIC.

The terms “wash buffer” or “post load wash”, as used herein, refer to abuffer used to elute one or more impurities from the ion exchange resinprior to eluting the protein of interest. The term “washing”, andgrammatical variations thereof, is used to describe the passing of anappropriate wash buffer through or over the chromatography resin.Conveniently, the wash, equilibration and loading buffers can be thesame, but this is not required. The pH and conductivity of the buffer issuch that one or more impurities are eluted from the resin while theresin retains the polypeptide of interest. If desirable, the wash buffermay contain a detergent, as described above, such as a polysorbate. Anysuitable buffer at a pH compatible with the selected resin can be usedfor purifying the protein of interest, such as the buffers describedabove. Selection of pH and conductivity of the wash buffer are importantfor removal of HCPs and other contaminants without significantly elutingthe protein of interest. The conductivity and pH can be reduced, ormaintained or increased in wash buffers used in subsequent wash stepsfor the HCIC and CEXC chromatography after loading the mixture in orderto remove more hydrophilic and more acidic or basic contaminants thanthat of the protein of interest and to reduce the conductivity of thesystem prior to the elution step. In a particular embodiment, only theconductivity is decreased for the HCIC chromatography, and post-loadwashes for CEXC do not include any change in either pH or conductivityof the buffers used for equilibration, load and post-load wash.

In a particular embodiment the wash buffer used for CEXC is sodiumphosphate. The pH of a wash buffer used in CEXC can be from about 3 toabout 9, more preferably pH from about 4.0 to about 8.0; andconductivity from about 0.1 to about 40 mS/cm, more preferablyconductivity from about 0.5 to about 10 mS/cm.

The wash buffer used in HCIC can be any suitable buffer to achieve adesirable pH and conductivity, and can be selected from a buffer asdescribed above. In a particular embodiment, the wash buffer used inHCIC is sodium phosphate. The pH of a wash buffer used in HCIC can befrom about 5 to about 10, more preferably pH from about 6 to about 9;and conductivity from about 0.0 (WFI) to about 90 mS/cm, more preferablyconductivity from about 0.1 to about 9 mS/cm.

The term “elution buffer”, as used herein, refers to a buffer used toelute the protein of interest from the solid phase (i.e, chromatographyresin). The term “elute”, and grammatical variations thereof, refers tothe removal of a molecule, e.g., polypeptide of interest, from achromatography material by using appropriate conditions, e.g., alteringthe ionic strength or pH of the buffer surrounding the chromatographymaterial, by addition of a competitive molecule for the ligand, byaltering the hydrophobicity of the molecule or by changing a chemicalproperty of the ligand (e.g. charge), such that the protein of interestis unable to bind the resin and is therefore eluted from thechromatography column. The term “eluate” refers to the effluent off thecolumn containing the polypeptide of interest when the elution isapplied onto the column. After elution of the polypeptide of interestthe column can be regenerated, sanitized and stored as needed.

The pH and conductivity of the elution buffer are selected such that theprotein of interest is eluted from the particular CEXC or HCIC resinused in the process. Buffers suitable for use as an elution buffer aredescribed above. In a particular embodiment, the elution buffer used inCEXC and HCIC chromatographies is sodium phosphate and sodium acetate,respectively.

The pH of the elution buffer used in CEXC can be from about 3.0 to about10, more preferably pH from about 4 to about 8; and conductivity fromabout 0.1 to about 40 mS/cm, more preferably conductivity from about 5to about 15 mS/cm.

The pH of the elution buffer used in HCIC can be from about 3.0 to about7.0, more preferably pH from about 4.0 to about 6.0; and conductivityfrom about 0.0 (WFI) to about 20 mS/cm, more preferably conductivityfrom about 0.1 to about 2 mS/cm.

If desired, additional solutions may be used to prepare the column forreuse. For example, a “regeneration solution” can be used to “strip” orremove tightly bound contaminants from a column used in the purificationprocess. Typically, the regeneration solution has a conductivity and pHsufficient to remove substantially any remaining impurities and proteinof interest from the resin.

As used herein, the term “conductivity” refers to the ability of anaqueous solution to conduct an electric current between two electrodesat a particular temperature. A current flows by ion transport insolution. Therefore, with an increasing amount of ions present in theaqueous solution, the solution will have a higher conductivity. In amethod of the present invention the temperature at which purification istypically performed can be from about 4 to about 37° C., more preferablyfrom about 15 to about 25° C. within the specified pH ranges.

The unit of measurement for conductivity is milliSiemens per centimeter(mS/cm), and can be measured using a standard conductivity meter. Theconductivity of a solution can be altered by changing the concentrationof ions therein. For example, the concentration of a buffering agentand/or concentration of a salt (e.g. NaCl or KCl) in the solution may bealtered in order to achieve the desired conductivity. Preferably, thesalt concentration is modified to achieve the desired conductivity asdescribed in the Example below.

The “pI” or “isoelectric point” of a polypeptide refers to the pH atwhich the polypeptide's positive charge balances its negative charge.The pI can be calculated according to various conventionalmethodologies, e.g., from the net charge of the amino acid and/or sialicacid residues on the polypeptide or by using isoelectric focusing.

As used herein, a “low pH hold” refers to a decrease in the pH of aneluate which contains the protein of interest, where the pH decrease isto less than about pH 5.0, preferably less than about pH 4.0, morepreferably less than about pH 3.7, and most preferably about pH 3.4 toabout 3.6 in order to achieve viral inactivation (i.e., at least a 2 logreduction in viral titer, more preferably at least a 3 log reduction)followed by an increase in pH in order to prepare the eluate for thesecond chromatography step or to bring the product to a matrix thatprovides stability to the molecular integrity of the product ofinterest. Any suitable acid can be applied to the eluate containing theprotein of interest to decrease the pH for the low pH hold, e.g., 1 NHCl or glacial acetic acid. Any suitable base can be applied to theeluate to return its pH to more neutral ranges, e.g., 1N NaOH or 1NTris. The low pH hold typically results in an increase in conductivityof at least 0.5 mS/cm. If the low pH hold takes place after the firstchromatography step and the second chromatography step is cationicexchange chromatography then it is likely that the increase in pH willbe to within a suitable pH range, which is about 3.0 to about 9.0,preferably about 4.0-8.0; If the low pH hold takes place after the firstchromatography step and the second chromatography step is HCIC then theincrease in pH will likely be within a pH range suitable for HCIC, whichis about pH 5 to about pH 10.0, preferably about pH 6.0 to about 9.0.The low pH hold can also take place after the second chromatography butin the method of invention has been chosen to take place in betweenchromatography steps due to experimental convenience. It shall beappreciated by those having ordinary skill in the art that a low pH holdstep is not essential to achieve levels of purity attainable by a methodof the present invention.

“Tangential flow filtration” or “TFF” or “crossflow filtration” refersto a filtration process in which the sample mixture circulates acrossthe top of a membrane, while applied pressure causes certain solutes andsmall molecules to pass through the membrane. In TFF, typically, thesolution flows parallel to the filter membrane. A pressure differentialacross the membrane causes fluid and filterable solutes (whose molecularweight is smaller than that of the membranes or behaves like so, such asglobular proteins) to flow through the filter. This can be conducted asa continuous-flow process, since the solution is passed repeatedly overthe membrane while the fluid that passes through the filter iscontinually drawn off into a separate circuit. In HPTFF (highperformance tangential flow filtration) the membrane is charged,therefore using both size and charge of molecules to separatecontaminants (see US Patent Pub. No. 2003/0229212). If desirable, TFFcan be used to exchange the buffer in which the protein of interest issolubilized into another buffer that is more suitable for binding ontothe chromatography resin prior to beginning a method of the presentinvention. In other words, TFF can be used before the firstchromatography step, however, an in-process TFF step (i.e., the TFFoccurs between the two chromatography steps) is unnecessary since thebuffers and resins used in the present method permit direct movementfrom one resin to the next.

As used herein, “in-process” refers to any methodology, step, operation,etc. that is performed after commencing the load step for the firstchromatography column, but prior to collection of the elution peak ofthe second chromatography column in a method of the present invention.An in-process methodology can directly result in a change in purity(such as an in-process TFF) or it does not directly result in a changein purity (such as a pH and/or conductivity adjustment where the resultof the adjustment does not directly cause any change in purity).

It may be desirable to achieve a therapeutic grade protein purity usinga method of the invention, in which case additional steps well-known inthe art can be optionally employed to achieve a viral clearance capacityfor both adventitious and endogenous viruses, e.g., low pH inactivationand viral filtration methods (including specific charged membranefiltration for viruses such as VR CUNO (CUNO) or DV20 (PallTechnologies), which are further described below.

As used herein “depth filtration” is a filtration method that uses depthfilters, which are typically characterized by their design to retainparticles within a filter matrix. The depth filter's capacity istypically defined by the depth, e.g. 10″ or 20″ of the matrix and thusthe holding capacity for solids. In a method of the present invention adepth filter can be used to improve the viral clearance capacity of thepurification scheme, however it is an optional step and not required toachieve purity levels of a method of the present invention. The depthfilter for virus removal can be used at any point during thepurification scheme, but is preferably used after the firstchromatography step with low process volumes, due to the cost of thefilter.

Description of the Process

In the method of the present invention CEXC and HCIC can be used in anyorder to purify a protein of interest to as little as about 100 ppm HCPor less and as high as 99% monomer purity or greater. Additionalchromatography steps are not needed to obtain this high level of purity.

The protein of interest can be produced by living host cells that havebeen genetically engineered to produce the protein. Methods ofgenetically engineering cells to produce proteins are well known in theart. See e.g. Ausabel et al., eds. (1990), Current Protocols inMolecular Biology (Wiley, New York) and U.S. Pat. Nos. 5,534,615 and4,816,567, each of which are specifically incorporated herein byreference. Such methods include introducing nucleic acids that encodeand allow expression of the protein into living host cells. These hostcells can be bacterial cells, fungal cells, or, preferably, animal cellsgrown in culture. Bacterial host cells include, but are not limited toE. coli cells. Examples of suitable E. coli strains include: HB101,DHSa, GM2929, JM109, KW251, NM538, NM539, and any E. coli strain thatfails to cleave foreign DNA. Fungal host cells that can be used include,but are not limited to, Saccharomyces cerevisiae, Pichia pastoris andAspergillus cells. A few examples of animal cell lines that can be usedare CHO, VERO, DXB11, BHK, HeLa, Cos, MDCK, 293, 3T3, NS0 and WI138. Newanimal cell lines can be established using methods well know by thoseskilled in the art (e.g., by transformation, viral infection, and/orselection). In particular embodiments, the protein of interest isproduced in a CHO cell (see, e.g., WO 94/11026). Various types of CHOcells are known in the art, e.g., CHO-K1, CHO-DG44, CHO-DXB11, CHO/dhfr⁻and CHO-S.

Preparation of a mixture for protein purification from cellular debrisinitially depends on the manner of expression of the protein. Someproteins are caused to be secreted directly from the cell into thesurrounding growth media, while other proteins are retainedintracellularly. For such proteins produced intracellularly, the cellcan be disrupted using any of a variety of methods, such as mechanicalshear, osmotic shock, and enzymatic treatment. The disruption releasesthe entire contents of the cell into the homogenate, and in additionproduces subcellular fragments which can be removed by centrifugation orby filtration. A similar problem arises, although to a lesser extent,with directly secreted proteins due to the natural death of cells andrelease of intracellular host cell proteins during the course of theprotein production run.

When using recombinant techniques, the protein of interest can beproduced intracellularly, in the periplasmic space, or directly secretedinto the medium. A method of the invention does not rely on anyparticular methodology to remove cellular debris. Any method can beemployed by the skilled practitioner to accomplish this. If the proteinis produced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, can be removed, for example, by acentrifugation or filtration step in order to prepare a mixture forpurification. If the protein is secreted into the medium, therecombinant host cells may be separated from the cell culture medium by,e.g., tangential flow filtration (TFF) or depth filtration, in order toprepare a mixture for purification.

According to the present invention, once a mixture containing theprotein of interest has been obtained, its separation from contaminantsin the mixture is performed using a combination of HCIC and CEXCchromatographies in either order as desired (see FIG. 1), underappropriate conditions, as described hereinabove. No other in-processpurification steps, e.g., TFF or HPTFF, are required to achieve levelsof purity attainable by a method of the present invention. Only pHmanipulations are performed as needed between the two chromatographysteps in order to prepare the mixture for purification in the secondstep of chromatography.

In a particular embodiment, the HCIC resin used is MEP HyperCel (PallCorp.) and the CEXC resin is Poros 50HS (Applied Biosystems). Thesechromatography techniques separate mixtures of proteins on the basis ofvarious properties, including charge and hydrophobicity. In essence,these separation methods cause proteins and other molecules to move atdifferent rates down a long column in order to achieve a physicalseparation as the material passes further down the column. Underappropriate conditions as described hereinabove, the chromatographysteps permit the protein of interest to adhere to the column, whileallowing other material to pass through the column. The protein ofinterest is then differentially eluted by appropriate buffer.Alternatively, under appropriate conditions, the desired protein can beseparated from impurities by adhering the impurities to the column,while the protein of interest passes through the column, i.e., theprotein of interest is present in the “flow-through.”

It shall be appreciated by those of ordinary skill in the art that a lowpH hold can be incorporated into the present method if desirable. Due toviral clearance requirements of regulatory agencies for the productionof therapeutic proteins, there must be at least two separate steps forviral inactivation and clearance, based on different chemical modes ofaction which are typically a low pH hold and a viral filtration step.The low pH hold typically employs pH shifts of the purified sample(e.g., see Example, infra.), and can be incorporated into the pHmanipulation necessary to prepare for the second chromatography step. Itshall be appreciated by those of ordinary skill in the art that a pHhold between the two chromatography steps can be accomplished using anymethodology known in the art to accomplish the viral clearance typicallyachieved by a low pH hold, provided that the mixture containing theprotein of interest is buffered appropriately prior to application onthe second column.

For example, without intending to be limiting, a low pH hold followingHCIC can be accomplished by using 1N HCl or glacial acetic acid todecrease the pH of the HCIC eluate to a pH range of about 3.4 to 3.6followed by 2M Tris pH 9.0 to increase the pH to of the eluate to about6.2; alternatively, following CEXC, 1N HCl or glacial acetic acid can beused to decrease the pH of the CEXC eluate to a pH range of about 3.4 to3.6 and 2M Tris pH 9.0 to increase the pH of the eluate to about 7.0. ApH hold will typically result in an increase of at least 0.5 mS/cm inconductivity.

One of ordinary skill in the art will appreciate timing conditions for alow pH hold step used in a method of the present invention. Typically, alow pH hold is performed for at least about 30 minutes, preferably about45 minutes, more preferably up to about 60 minutes, and most preferablyup to about 90 minutes. In certain purification schemes it may bedesirable to perform a low pH hold for up to 5 hours depending on theprotein being purified

If therapeutic grade protein formulation is desirable, then a secondviral clearance step can be employed, e.g., a virus filtration step,which is, however, not required to achieve the levels of purityattainable by a method of the invention. Filtration devices that can beused in a method of the invention are well-known in the art (e.g.,Ultipor® VF Grade DV20 or DV50 and Filtron® TFF (Pall Corporation, EastHills, N.Y.); Viresolve (Millipore, Billerica, Mass.); VR CUNO (CUNO;Meriden, Conn.); and Planova® (Asahi Kasei Pharma, Planova Division,Buffalo Grove, Ill.), in order to remove viruses and other biologicalmaterials, preferably with pore sizes smaller than 20 nm, which canremove polioviruses.

The chromatographic steps of the invention can be carried out by anymechanical means. Chromatography may be carried out in a column. Thecolumn may be run with or without pressure and from top to bottom orbottom to top. The direction of the flow of fluid in the column may bereversed during the chromatography process. Chromatography may also becarried out using a batch process in which the solid support isseparated from the liquid used to load, wash, and elute the sample byany suitable means, including gravity, centrifugation, or filtration.Chromatography may also be carried out by contacting the sample with acharged filter that absorbs or retains some molecules in the sample morestrongly than others, using the same chemical principles as thosedescribed for a chromatography resin. Although steps in the preparationof columns used in the present invention can be tailored to suit theindividual needs of the practitioner, the following description isprovided as guidance, and those having ordinary skill in the art willappreciate alterations can be made without departing from the spirit ofthe invention. Columns are prepared according to the manufacturer'sinstructions. Prior to purification, columns are typically sanitizedusing a sanitization solution, then charged using a lyotropic salt,e.g., 1 M NaCl, and equilibrated using an equilibration buffer. Duringthe sanitization step, typically there is a hold, e.g., for about 30minutes, preferably for about 1 hour, after the sanitization solution isapplied onto the column in order to clean the resin having any boundcontaminants, including contaminants of biological origin. The chargestep neutralizes the resin by displacing the sanitization solution,e.g., in CEXC, NaCl can be used to remove NaOH from the column andmaintain the resin ligand in contact with positively charged ions. Afterthe column is charged, an equilibration buffer is used to equilibratethe column in order to prepare the pH and conductivity of the resin tobind the protein of interest. For example, the column can be consideredequilibrated when its pH and conductivity are within ±0.1 and within±0.2 mS/cm of the pH and conductivity of the equilibrating buffer,respectively.

A load mixture is prepared, which is typically a cell culturesupernatant that is concentrated and buffer exchanged in an appropriatebuffered salt (i.e. load buffer). The load mixture (i.e., containing theprotein of interest for which purification is desired) derived from therecombinant host cells is loaded onto the equilibrated column (eitherCEXC or HCIC) using a loading buffer which optionally can be the same asthe equilibration buffer. As the mixture flows through the solid phaseof the column, the protein of interest and other impurities (e.g., HCPsif the protein is produced in a CHO cell) bind differentially to thesolid phase, thereby permitting separation as the protein andcontaminants pass through the chromatography column.

Once the mixture has been loaded onto the column and is bound to theresin, wash steps using a wash buffer as described are performed toclear contaminants from the column. Optionally, a first wash step can beperformed at a slower flow rate than for the other wash and elutionsteps (but is not necessary), e.g., using a flow rate corresponding toseveral minutes (e.g., 5-10 minutes) residence time. Flow rates arediscussed in greater detail infra. The pH and conductivity of the washbuffer are important during the first wash, particularly for HCICchromatography, in order to maximize removal of HCPs. In the presentmethod, the wash step in CEXC chromatography removes nucleic acids andremaining HCPs while retaining the protein of interest. The first washstep in HCIC chromatography removes HCPs while the second wash removesmore hydrophilic contaminants, e.g., HCPs. HCIC is very efficient fornucleic acid removal because it mostly flows in the unbound fraction. IfHCIC is employed as the first column, then a third wash step can beemployed to further reduce levels of any hydrophilic contaminants.

To elute the protein of interest from the column, an appropriate elutionbuffer is used, as described above, to cause the protein of interest toreverse binding off the column. The type and concentration of buffer,salt, and/or other compound in the buffer composition are such that thebuffer elutes impurity(ies) differentially in relation to the protein ofinterest, whereas the protein of interest is retained relative to theimpurities. The appropriate pH and conductivity ranges for loading,wash, and elution buffers can be readily determined by those havingordinary skill in the art, using the Example provided herein asguidance, such that the protein of interest is recovered during elution.In order to minimize HCPs in the eluate containing the protein ofinterest, a reduced flow rate, described infra., can be employed, e.g.,not greater than a corresponding residence time of 2 minutes, morepreferably not greater that 3 minutes residence time, more preferably nogreater than 10 minutes residence time. As the protein of interest isremoved from the column, it is collected based on formation of a peakfrom rising A₂₈₀ to falling A₂₈₀ between 5 and 25% above baseline, asmeasured by absorbance. The baseline can be readily determined by one ofordinary skill in the art by measuring the absorbance of theequilibrating buffer at 250-280 nm while the buffer passes through thecolumn. In order to reduce collection of HCPs to prepare a purifiedcomposition containing the protein of interest, it is preferred thateluate containing the protein of interest is collected before 25% ofmaximum peak height, since HCPs tend to elute at a later time or atlower pH values for the HCIC resin.

In essence, the separation methods used in the present invention causecontaminants to move at different rates down a long column, achieving aphysical separation that increases as they pass further down the column,while the protein of interest adheres to the chromatography medium, andis then differentially eluted depending on the buffer. In the presentpurification method, the maximum rate at which a mixture moves down thecolumn (HCIC or CEXC) is not greater than 30 minutes residence time,preferably not greater than 10, more preferably not greater than 6minutes, still more preferably not greater than 3 minutes, and mostpreferably not greater than 2 minutes. In a particular embodiment of theinvention, the flow rate in the CEXC step corresponds to a residencetime not greater than about 2 minutes; in the HCIC step it is notgreater than 3 minutes. While the flow rate is not essential toachieving purity levels obtainable by a method of the invention, ratesare provided for purposes of guidance. One having ordinary skill in theart can modify the flow rate as needed.

The preferred measure of protein purification by a process of thepresent invention is the measure of host cell protein removal. Thepurified protein is preferably a homogenous composition as it is definedsupra. Protein concentration of a sample at any stage of purificationcan be determined by any suitable method. Such methods are well known inthe art and include: 1) colorimetric methods such as the Lowry assay,the Bradford assay, the Smith assay, and the colloidal gold assay; 2)methods utilizing the UV absorption properties of proteins; and 3)visual estimation based on stained protein bands on gels relying oncomparison with protein standards of known quantity on the same gel. Seee.g. Stoschek (1990), Quantitation of Protein, in Guide to ProteinPurification, Methods in Enzymol. 182: 50-68. Protein contamination of ahomogenous composition containing the protein of interest can bedetermined by various means know in the art, e.g., by enzyme-linkedimmunosorbent assay (ELISA; see e.g. Reen (1994), Enzyme-LinkedImmunosorbent Assay (ELISA), in Basic Protein and Peptide Protocols,Methods Mol. Biol. 32: 461-466, which is incorporated herein byreference in its entirety).

The amount of DNA that may be present in a homogenous compositioncontaining the protein of interest can be determined by any suitablemethod. For example, one can use an assay utilizing polymerase chainreaction. DNA can be reduced to levels that are lower than 10 pg/mg.

The protein thus recovered can be formulated in a biopharmaceuticallyacceptable composition and is used for various diagnostic, therapeuticor other uses known for such molecules.

After collection of the eluate containing the protein of interest, anyproteins that may remain bound to the column can be released bystripping the chromatography medium using a solution comprising thebuffer or salt used for chromatography, but at a higher molarity ormodified pH. The column may then be regenerated using a solution (e.g.,a regeneration solution) that will have the effect of releasing most orall proteins from the chromatography medium and reducing or eliminatingany microbial contamination that may occur in the chromatography medium.Subsequently, the column may be rinsed and stored in a solution thatdiscourages microbial growth. Such a solution may comprise sodiumhydroxide, but other reagents can also be used, such as ethanol, sodiumazide, benzyl alcohol or high concentration of lyotropic salts.

The following Example is provided for purposes of demonstrating theinvention, and is not intended to be limiting.

Example

The present Example describes the purification of antibodies using apurification method of the present invention. Fully human antibodieswere prepared against an activated T cell associated antigen (CTLA-4)and a tumor associated antigen (CD30) using Medarex's HuMAb Mouse®.Transfectomas were prepared using CHO DG44 cells. Cell culture wasperformed using synthetic, serum-free, defined medium at neutral pH (7to 8) and conductivity between 10 and 16 mS/cm. The DG44 CHO cells weregrown to a density of about 3 to 10×10⁶ cells/ml. Cell culture wasclarified by filtration using a 60MO2 CUNO filter (Meriden, Conn.). Theresulting cell culture supernatant was concentrated and diafiltered ineither 35 or 70 mM Sodium Phosphate, pH 6.2. For experiments where MEPHypercel was used as the capture resin, the load was adjusted to pH 7 bytitrating the concentrated and diafiltered cell culture supernatant with1M NaOH. Purification of mixtures containing either of the twoantibodies was performed in either direction as described below.

Tables 1 and 2 show examples of the two steps in a purification processof the invention for production of therapeutic grade protein material.In this process (A), the first non-affinity CEXC chromatography step wascationic exchange chromatography (Table 1) using Poros 50HS resin forcapture, followed by a low pH hold using 1N HCl or glacial acetic acidto decrease the pH of the eluate to 3.4 to 3.6 and 2M Tris pH 9.0 toincrease the pH of the fraction to 7.0. The second non-affinity step wasHCIC (Table 2) using MEP HyperCel resin for capture. The low pH holdtypically resulted in an increase of about 2mS/cm in conductivity. Theprocess was carried out using equilibration, loading, wash and elutionbuffers for the respective resin as indicated in Tables 1 and 2. Neitherpurification step used an in-process buffer exchange step, i.e., thesample proceeded from one chromatography step to the next with only pHmanipulations as part of the low pH hold.

An alternate process (B) for production of therapeutic grade proteinmaterial was performed where the CEXC and HCIC chromatography steps werereversed, i.e., the steps in Table 2 were performed first followed bythe steps in Table 1. The first non-affinity chromatography step wasHCIC using MEP HyperCel resin for capture, as described in Table 2. Alow pH hold using 1N HCl or glacial acetic acid was performed todecrease the pH of the MEP eluate to 3.4 to 3.6 followed by 2M Tris, pH9.0 to increase the pH of the fraction to 6.2. The low pH hold typicallyresulted in an increase of about 2mS/cm in conductivity. The second(CEXC) chromatography step was CEXC using Poros 50HS resin forpolishing, as described in Table 1. The process was carried out usingequilibration, loading, wash and elution buffers for the respectiveresin as indicated in the Tables. None of the purification steps in thealternate process included an in-process buffer exchange step, i.e., thesample proceeded from one chromatography step to the next with only pHmanipulations as part of the low pH hold.

TABLE 1 Cation exchange chromatography for antibody capture orpolishing: summary of major operational details. Maximum Residence Cond.Vol. Time Step Solution pH (mS/cm) (CVs) (minutes) Forward ProcessingCriteria Sanitization 1N NaOH ≧12 N/A ≧3 2 Usually there is a hold ofone hour, starting from the time the solution is applied onto thecolumn. This step intends to clean the resin from bound contaminants,including those of biological origin. Charge 1M NaCl N/A ~90 ≧3 2 NaClremoves NaOH from the column, while neutralizing and maintaining theligand of the resin in connection with sodium ions. Equilibration 35 or6.2 ~3 for the ≧3 2 Column is equilibrated when pH and conductivity 70mM NaP 35 mM NaP are ±0.1 and ±0.2 mS/cm of the equilibrating ~5.8 forthe buffer, respectively. Conductivity and pH ranges 70 mM NaP areimportant to bind the antibody, while minimizing the binding of hostcell proteins. The highest concentration without compromising bindingcapacity should be used. Load Product 6.2 ~1 for MEP 2 Load is cellculture supernatant, concentrated eluate and buffer exchanged in 35 or70 mM Sodium ~3 for the Phosphate, pH 6.2, in accordance with the 35 mMNaP equilibration buffer, as CAPTURE. Before sterile load for anti-filtration, the product can be filtered through CD30 Ab a CUNO membrane.As a POLISH, load is MEP HyperCel ~5.8 for the eluate that underwent thelow pH hold. 70 mM NaP load for anti- CTLA4 Ab Post Load 35 mM NaP 6.2~3 for the ≧3 2 Slower flow rate of the buffer containing Wash 1 with0.1% 35 mM NaP Polysorbate 80 helps to reduce the DNA Tween 80 contentof the final purified product. Post Load 35 mM NaP 6.2 3-5.8 ≧7 2 Columnis equilibrated when pH and conductivity Wash 2 are ±0.1 ±0.2 mS/cm ofthe equilibrating buffer. Conductivity and pH ranges are important tobind the antibody, while minimizing the binding of host cell proteins.Elution 35 mM NaP 6.2 ~7 for 35 mM  ~5 2 Collect the elution peak fromrising Abs₂₈₀ to 40-75 mM NaP, 40 mM falling Abs₂₈₀ 20% above baseline.Do NOT collect NaCl NaCl for anti- the tailing of the peak to ensurelower CHOP level CD30 Ab in elution. ~10.5 for 35 mM NaP, 7.5 mM NaClfor anti- CTLA4 Ab Strip 1M NaCl N/A 69.1-95.9 ≧5 2 Removes tightlybound contaminants. CIP 1N NaOH ≧12 N/A ≧3 2 Similar step tosanitization. Storage 0.1N ≧12 N/A ≧3 2 Alkalinity of this solution doesnot allow NaOH microbial growth but maintains resin integrity. Bindingcapacity was constant at 15 mg/ml resin. Flow rates are expressed incorresponding residence times to provide wider experimental flexibility.Working temperature varied between 15-25° C. NaP = sodium phosphate.

TABLE 2 MEP HyperCel chromatography for antibody capture or polishing:summary of major details. Maximum Residence Cond. Vol. Time StepSolution pH (mS/cm) (CVs) (minutes) Forward Processing CriteriaSanitization 1N NaOH ≧12 N/A ≧3 3 Usually there is a hold of one hour,starting from the time the solution is applied onto the column. Thisstep intends to clean the resin from bound contaminants, including thoseof biological origin. Charge 1M NaCl N/A ~90 ≧3 2 NaCl removes NaOH fromthe column, while neutralizing and maintaining the ligand of the resinin connection with sodium ions. Equilibration 35 or 7.0 ~4 (35 mM NaP)≧3 3 Column is equilibrated when pH and conductivity 70 mM NaP ~8 (70 mMNaP) are ±0.1 and ±0.2 mS/cm of the equilibrating buffer, respectively.Load Product 7.0 ~4 for the 3 Load is cell culture supernatant,concentrated and 35 mM NaP used buffer exchanged in 35 or 70 mM SodiumPhosphate, in capture for pH 6.2, and adjusted to pH 7.0, as CAPTURE.anit-CD30 Ab Before sterile filtration, the product can be ~8 for thefiltered through a CUNO membrane. As a POLISH, 70 mM NaP used load isCEXC eluate that underwent a low pH hold. in capture for anti-CTLA4 Ab~10 for the CEX eluate used in polishing Post Load 70 mM NaP 7.0 ~8 ≧5 3Optional Wash 1 Post Load 35 mM NaP 7.0 ~4 ≧5 3 pH and conductivity ofthese buffers are very Wash 2 crucial for contaminants (CHOP) removal.Post Load 5 mM NaP 7.0 <1 ≧5 3 Lower conductivity of the buffer removesmore Wash 3 hydrophilic contaminants and reduces the conductivity of thesystem, prior to elution. Elution 10 mM Na 5.2 <1 ~3 to 5 3 to 6 Collectthe elution peak from rising A₂₈₀ to Acetate falling A₂₈₀ 20% abovebaseline. Do NOT collect the tailing of the peak to ensure lower CHOPlevel in elution. Slow flow rate minimizes the host cell protein in theelution. Strip 100 mM 3.0 ~4 ≧5 3 Removes tightly bound contaminants.NaCitrate CIP 1N NaOH ≧12 N/A ≧3 3 Similar step to sanitization. Storage0.1N ≧12 N/A ≧3 3 Alkalinity of this solution does not allow NaOHmicrobial growth but maintains resin integrity. Binding capacity wasconstant at 15 mg/ml resin. Flow rates are expressed in correspondingresidence times to provide wider experimental flexibility. Workingtemperature varied between 15-25° C. NaP = sodium phosphate.

Using a standard ELISA assay to confirm CHOP levels, PCR to confirm DNAlevels, and HPLC-SEC to confirm the monomer purity of the antibody, eachof the two purification procedures A and B, achieved a homogenouscomposition, containing less than 100 ppm host cell protein (see Table 3below), less than 10 pg DNA/mg antibody and greater than 99% monomer,which permits further processing and formulation of the purifiedmaterial to a therapeutic grade product. The contaminant concentrationafter the first step varied between 200 and 1500 ng/mg for CHOPs and 95to 100% monomer. The results for processes A and B using two differentfully human monoclonal antibodies are reported in Table 3.

TABLE 3 Summary of contaminant removal using suggested purificationschemes. Process Parameter (anti-CTLA-4 Ab) (anti-CD30 Ab) CEXC→HCIC %Overall Recovery 74 68 Final CHOP (ng/mg) 30.8 44.2 Final DNA (pg/mg)<10.00 <10.00 Final Purity (% Monomer by HPLC-SEC) 100.00 99.55HCIC→CEXC Overall Recovery 84.8 74.5 Final CHOP (ng/mg) 69.9 42.6 FinalDNA (pg/mg) <10.00 <10.00 Final Purity (% Monomer by HPLC-SEC) 100.0099.82

1. A method for purifying a target protein from a mixture, whichcomprises the target protein and one or more contaminants, comprising:(a) subjecting the mixture to a cation exchange chromatography step anda hydrophobic charge induction chromatography step, in either order,wherein there is no in-process tangential flow filtration step; and (b)isolating the target protein.
 2. The method of claim 1, wherein themixture is subjected to the hydrophobic charge induction chromatographystep first.
 3. The method of claim 1, wherein the mixture is subjectedto the cation exchange chromatography step first.
 4. The method claim 1,wherein the target protein is isolated to a purity of about 100 partsper million (ppm) or less of host cell protein and about 10 pg/mg orless of nucleic acids.
 5. The method of claim 1, which further includesa viral inactivation step.
 6. The method of claim 5, wherein the viralinactivation step is an in-process step.
 7. The method of claim 1,wherein the mixture is prepared from a cell culture supernatant.
 8. Themethod of claim 1, wherein the cation exchange chromatography isperformed at a pH range from 3 to
 10. 9. The method of claim 1, whereinthe cation exchange chromatography is performed at a pH range from 4.0to 8.0.
 10. The method of claim 1, wherein the hydrophobic chargeinduction chromatography step is performed at a pH range from 3 to 10.11. The method of claim 1, wherein the hydrophobic charge inductionchromatography is performed using at a pH range from 4.0 to 9.0.
 12. Themethod of claim 1, wherein the target protein binds to a cation exchangechromatography column at pH 3 to 9 and at a conductivity range from 0.1to 40 mS/cm.
 13. The method of claim 1, wherein the target protein bindsto a cation exchange chromatography column at pH 4.0 to 8.0 and at aconductivity from 0.5 to 10 mS/cm.
 14. The method of claim 1, whereinthe target protein binds to a hydrophobic charge inductionchromatography column at pH 5 to 10 and at a conductivity range from 0to 90 mS/cm.
 15. The method of claim 1, wherein the target protein bindsto a hydrophobic charge induction chromatography column at pH 6 to 9 andconductivity range from 2 to 9 mS/cm.
 16. The method of claim 1, whereinthe target protein is bound to the cation exchange chromatography columnand the column is washed using a wash buffer having a pH range from 3 to9 and a conductivity range from 0.1 to 40 mS/cm.
 17. The method of claim1, wherein the target protein is bound to a cation exchangechromatography column and the column is washed using a wash bufferhaving a pH range from 4.0 to 8.0 and a conductivity range from 0.5 to10 mS/cm.
 18. The method of claim 1, wherein the target protein is boundto a hydrophobic charge induction chromatography column and the columnis washed using a wash buffer having a pH range from 5 to 10 and aconductivity range from 0 to 90 mS/cm.
 19. The method of claim 1,wherein the target protein is bound to a hydrophobic charge inductionchromatography column and the column is washed using a wash bufferhaving a pH range from 6 to 9 and a conductivity range from 0.1 to 9mS/cm.
 20. The method of claim 1, wherein the target protein elutes fromthe cation exchange chromatography column at a pH range from 3 to 10 anda conductivity range from 0.1 to 40 mS/cm.
 21. The method of claim 1,wherein the target protein elutes from the cation exchangechromatography column at a pH range from 4.0 to 8.0 and a conductivityrange from 5 to 15 mS/cm.
 22. The method of claim 1, wherein the targetprotein elutes from the hydrophobic charge induction exchangechromatography column at a pH range from 3.0 to 7.0 and a conductivityrange from 0 to 20 mS/cm.
 23. The method of claim 1, wherein the targetprotein elutes from the hydrophobic charge induction exchangechromatography column at a pH range from 4.0 to 6.0 and a conductivityrange from 0.1 to 2.0 mS/cm.
 24. The method of claim 1, wherein thecation exchange chromatography step employs a cation exchange ligandselected from sulfonate, carboxylic, carboxymethyl sulfonic acid,sulfoisobutyl, sulfoethyl, carboxyl, sulphopropyl, sulphonyl,sulphoxyethyl and orthophosphate.
 25. The method of claim 1, wherein thecation exchange chromatography step is performed on a cation exchangeresin selected from Poros HS, Poros S, carboxy-methyl-cellulose,BAKERBOND ABX™, sulphopropyl immobilized on agarose and sulphonylimmobilized on agarose, MonoS, MiniS, Source 15S, 30S, SP sepharose, CMSepharose, BAKERBOND Carboxy-Sulfon, WP CBX, WP Sulfonic, Hydrocell CM,Hydrocel SP, UNOsphere S, Macro-Prep High S, Macro-Prep CM, CeramicHyperD S, Ceramic HyperD CM, Ceramic HyperD Z, Trisacryl M CM, TrisacrylLS CM, Trisacryl M SP, Trisacryl LS SP, Spherodex LS SP, DOWEX Fine MeshStrong Acid Cation Resin, DOWEX MAC-3, Matrex Cellufine C500, MatrexCellufine C200, Fractogel EMD SO3—, Fractogel EMD SE, Fractogel EMDCOO—, Amberlite Weak and Strong Cation Exchangers, Diaion Weak andStrong Cation Exchangers, TSK Gel SP-5PW-HR, TSK Gel SP-5PW, ToyopearlCM (650S, 650M, 650C), Toyopearl SP (650S, 650M, 650C), CM (23, 32, 52),SE(52, 53), P11, Express-Ion C and Express-Ion S.
 26. The method ofclaim 1, wherein the hydrophobic charge induction chromatography stepemploys a hydrophobic charge induction chromatography ligand comprisingan ionizable aromatic or heterocyclic structure which has an aliphaticchain and at least one sulfur atom on the linker arm and/or ligandstructure.
 27. The method of claim 26 wherein the hydrophobic chargeinduction chromatography ligand comprises a group selected from2-aminomethylpyridine, 3-aminomethylpyridine and 4-aminomethylpyridine,2-mercaptopyridine, 4-mercaptopyridine or 4-mercaptoethylpyridine,mercaptoacids, mercaptoalcohols, imidazolyl based,mercaptomethylimidazole, 2-mercaptobenzimidazole,aminomethylbenzimidazole, histamine, mercaptobenzimidazole,diethylaminopropylamine, aminopropylmorpholine, aminopropylimidazole,aminocaproic acid, nitrohydroxybenzoic acid, nitrotyrosine/ethanolamine,dichlorosalicylic acid, dibromotyramine, chlorohydroxyphenylacetic acid,hydroxyphenylacetic acid, tyramine, thiophenol, glutathione, bisulphate,and dyes, or a derivative thereof.
 28. The method of claim 1, whereinthe hydrophobic charge induction chromatography step is performed usinga hydrophobic charge induction resin selected from MEP HyperCel.
 29. Themethod of claim 1, wherein the contaminants are selected from host cellproteins, host cell metabolites, host cell constitutive proteins,nucleic acids, endotoxins, product related contaminants, lipids, andmedia additives or media derivatives.
 30. The method of claim 1, whereinthe target protein is an antibody or antibody fragment.
 31. The methodof claim 30 wherein the antibody is a monoclonal antibody.
 32. Themethod of claim 30 wherein the antibody is a fully human antibody. 33.The method of claim 30 wherein the antibody is selected from anti-CTLA4antibody and anti-CD30 antibody.
 34. The method of claim 30 wherein theantibody is selected from single-chain antibody molecules, diabodies,linear antibodies, bispecific antibodies and multispecific antibodies.35. The method of claim 30 wherein the antibody fragment is selectedfrom Fab, Fab′, F(ab′)₂ and Fv.
 36. The method of claim 1, wherein thetarget protein is an immunoadhesin.
 37. The method of claim 1, furthercomprising the step of formulating the isolated protein into apharmaceutical composition.